FAO Symposium on

“The role of agricultural biotechnologies for production of bio-energy in

developing countries"

ETHANOL PRODUCTION VIA ENZYMATIC

HYDROLYSIS OF SUGAR-CANE BAGASSE AND STRAW

Elba P. S. Bon
Chemistry Institute
Federal University of Rio de Janeiro - Brazil
elba1996@iq.ufrj.br
 Presentation Outline

• Overview of the Current Brazilian Ethanol Production

• Biomass Ethanol

• The BIO-ETHANOL Project - Ethanol Production from

Sugarcane Biomass via Enzymatic Hydrolysis
 Main Crops in Brazil
Brasil: 851 x 106 hectares

Area
[106 ha]
Ceará: 14,6 106 ha
●
●
Paraíba: 5,7 106 ha
Soya 21,5

Corn 12,3

Sugar cane 5,6

● Paraná: 20,0 106 ha
 SUGARCANE / ETHANOL PRODUCTION

Brazil has 365 sugar/ethanol producing units from which 240

produce both sugar and ethanol, 109 produce only ethanol
and 15 produce only sugar.

It is forecast that 41 new distilleries will be built until 2010

70,000 farmers produced 428 million tons of sugarcane

(2006/2007 harvest)

The amount of dry bagasse annually produced by the ethanol

and sugar industry is of 64 million tons

Refineries made 4 billion gallons of alcohol fuel.

Ethanol production replaces 460 million barrels of oil

 Sugar-cane Plantation

Harvest
Non mechanized →Straw
Mechanized→
ETHANOL FROM AGROINDUSTRIAL RESIDUE

Straw

and/or

Bagasse

↓
ETHANOL
 Sugar Cane Bagasse Composition

Cellulose →C 6 Sugars for ETHANOL

~37% (Renewable liquid fuel)

Hemicellulose →C 5 Sugars for BIO-REFINERIES

~28% or ENZYMES PRODUCTION

Lignin → Poly aromatic hydrophobic

~21% structure
(Renewable Solid Fuel)
 Presentation Outline

• Overview of Ethanol Production from Sugar-cane

(sucrose)

• Biomass Ethanol

• The BIO-ETHANOL Project - Ethanol Production

from Sugar-cane Biomass
No longer oil....biomass!
CELLULAR STRUCTURE OF BIOMASS
1 mm
SEM

Source: Himmel et.al.

in collaboration
with the CSM EM Facility
(2004)
 Cell wall structure
Buffer treated corn stover Enzyme treated corn stover

Enzymatic hydrolysis
of cellulose to
glucose

Note: zone around vascular bundle is eroded compared to native

(suggests enzymes leak through pores in bundle)
 CELL WALL STRUCTURE

1st Challenge: Cell W all Recalcitrance

• Lignocellulose cell walls
contain interm eshed
carbohydrate and lignin
polym ers and other
m inor constituents
– The major structural
polymers – cellulose,
hemicellulose, and
lignin – exhibit
differential reactivity to
thermal, chemical, and
biological processing
– By natural design, cell
wall polysaccharides
are more difficult to
break down than
storage carbohydrates
like starch
CELL WALL STRUCTURE

Cellulose
Hemicellulose
Lignin
 Bioconversion of cellulose to ethanol

• Pre-treatment

• Cellulases Production

• Enzymatic Hydrolysis

• Ethanol Fermentation
Enzymes: Endoglucanases e Exoglucanases
 Presentation Outline

• Overview of the Current Ethanol Production:

Sucrose Sugarcane

• Biomass Ethanol

• The BIOETHANOL Project - Ethanol Production

from Sugar-cane Biomass
 Bioethanol Project - Synopsis

A RESEARCH NETWORK has been organized to develop

in Brazil the technology for the conversion of the
sugarcane biomass (bagasse and straw) into fuel
ethanol. The hydrolysis of the lignocellulose biomass to
release the C6 fermentable sugars will be carried out via
enzymatic hydrolysis.
 Brazilian Institutions / International Collaboration

• 14 Brazilian Universities: UNICAMP/Campinas, USP/Lorena,

USP/SãoPaulo, UFRJ/Rio de Janeiro, UENF/Campos, UEM/Maringá,
UnB/Brasília, UCS/Caxias do Sul, FURB/Blumenau, UFPE/Recife,
UFPB/JoãoPessoa),UFPR/Curitiba,UFG/Goiânia, UFSc/Florianópolis
• 2 Research Centers: (IPT/São Paulo and INT/Rio de Janeiro)
• Center for Sugarcane Technology Development - CTC/Piracicaba
(congregates around 100 sugar and ethanol industries)
• CENBIO (National Centre for Biomass Development)/São Paulo

Foreign Collaboration
• LUND UNIVERSITY - Sweden
• The project is open to the establishment of new collaborations.
Main Research Areas
• Development of biomass pre-treatment processes for
sugar cane bagasse and straw
• Raw and pre-treated biomass characterization
• Cellulases / xylanases production
• Enzymatic hydrolysis
• Sugars syrups characterization
• Ethanol fermentation (C6)
• C5 sugars and lignin uses
• Energy optimisation
• Effluents and water
 ETHANOL FROM BIOMASS
Bagasse or Straw

Pentoses (hemicellulose)
Pre-treatment
Bio-Refineries/Enzyme Production

Lignin
Enzyme Enzymatic Hydrolysis (Solid Residue)

GLUCOSE

Sucrose Ethanol

Sucrose Juice or Ethanol Fermentation Yeast

Molasses

Distillation Stillage

Ethanol
Steam Pre-treatment
Steam Pre-treatment
Sugar-cane Bagasse

Sugar-cane Bagasse Treated Sugar-cane Bagasse

 Enzymes Production for
Biomass Ethanol - Principles

Enzyme cost contribution and effectiveness depends

on the biomass source and pre-treatment
conditions.

Development of “tailored made” enzyme blends for
sugar-cane biomass.

Use of crude “cellulase/xylanase/accessory
enzymes” preparations.

“In house” production to reduce the enzymes cost.

Cellulases production
Cellulolytic enzymes are mainly produced by various microorganisms:

Aerobic bacteria
Anaerobic bacteria
Fungi

Actinomycetes
Clostridium

Trichoderma

Aspergillus
Studies on Cellulolytic/Xylanolitic Enzymes

• Enzyme production using fed batch fermentation

• Enzyme production using semi-solid fermentation
• Production and use of inducers (sophorose)
• Selected genes cloning and expression
• Microorg. and genes identification and preservation
• Culture supernatants concentration
• Enzyme activity stabilization
• Evaluation of the effectiveness of different enzyme blends
• Enzymes chemical and biochemical characterization
Cellulases production

BIOETHANOL cellulases
blend
Cellulase production
Enzymes Production and Uses
Industrial application of cellulases

The major challenge: to convert,

economically, biomass residues
into glucose !
 Enzymatic Hydrolysis Experiments

130 g/L of treated

sugar-cane bagasse

Enzyme blend

Sodium Citrate Buffer

pH 4.8

Temperature 45ºC

Agitation 200 rpm

 Enzymatic Hydrolysis Kinetics

Bagasse concentration = 130 g/L (dry weight)

% cellulose in bagasse = 55%

Cellulose hydrolysis yield using commercial enzyme: 61%

Cellulose hydrolysis yield using BIOETHANOL enzyme = 90%

70
60
BIOETHANOL ENZYME
Glucose (g/L)

50
40 COMMERCIAL ENZYME
30
20
10
0
0 20 40 60 80
Time (h)
 Sugar Syrups and Lignin

Glucose Syrup
(65 g/L)

Ethanol
fermentation

Residue: Lignin
Treated bagasse
+ BIOETHANOL Enzymes
Burning for Energy
 Lignocellulosic hydrolysate

Almeida et. al. (2007). J Chem. Technol. & Biotechnol 82: 340-349
 Effects of lignocellulosic inhibitors on the yeast
cells (Saccharomyces cerevisiae)

Almeida et. al. (2007). J Chem. Technol. & Biotechnol 82: 340-349
 Ethanol fermentation

Saccharomyces cerevisiae industrial strain

Hydrolyzate sugar syrup – 50 g/L glucose

Inocullum: 200 mg cell dry weight/ 15 mL

Experiments:

Growth medium (400mM total sugar)

Biomass sugar syrup
1. Sugar mollases
2. Yeast extract dextrose
Ethanol fermentation

Ethanol
quantification
 BAGASSE PRODUCTION AND
AVAILABILITY/YEAR (KG)

Total sugarcane production 428.000.000.000

Total bagasse production (30% of 128.400.000.000
sugarcane - 50% moisture)
Total dry bagasse production 64.200.000.000
Surplus bagasse (12% dry bagasse) 7.700.000.000

THEORETICAL ETHANOL YIELD FROM SUGAR-

CANE BIOMASS CELLULOSE

1 Kg of bagasse: 0,24L
7.700.000.000 Kg of bagasse: 1.849.000.000 L
 Enzyme cost contribution

Enzyme load: 10 FPU (filter paper unit) per gram of bagasse

Industrial Enzyme: 140.000 FPU/L

Enzyme price Enzymes cost Enzyme market

(US$/L) contribution per L of (US$/year)
ethanol (US$/L)
7,00 2,03 2.290.000.000
(FOB price)

0,17 0,05 * 56.400.000

* Target enzyme cost contribution

 Summary of results: BIOETHANOL Project

Environmental friendly steam based pre-

treatment (no use of chemicals)

Efficient BIOETHANOL enzyme blend

Biomass hydrolysate in high sugar yields –
concentration will be improved further

The C6 sugars syrup are fermentable by
usual industrial Saccharomyces cerevisiae
strain (no GMO)

The syrup composition favors ethanol yield
 On going work

Pilot Plant

• Biomass steam pretreatment

• “In house” enzymes production
• Enzymatic biomass hydrolysis
• Syrup blend to sugar cane juice or molasses
• Current fermentation process
• Use of lignin as fuel
The historically old Saccharomyces cerevisiae!

BIOREFINERIES
BIOETHANOL RESEARCHERS
Thank You!